The granular dorsolateral prefrontal cortex (dlPFC) is an evolutionary specialization of primates that is centrally involved in cognition. We assessed more than 600,000 single-nucleus transcriptomes from adult human, chimpanzee, macaque, and marmoset dlPFC. Although most cell subtypes defined transcriptomically are conserved, we detected several that exist only in a subset of species as well as substantial species-specific molecular differences across homologous neuronal, glial, and non-neural subtypes. The latter are exemplified by human-specific switching between expression of the neuropeptide somatostatin and tyrosine hydroxylase, the rate-limiting enzyme in dopamine production in certain interneurons. The above molecular differences are also illustrated by expression of the neuropsychiatric risk gene FOXP2, which is human-specific in microglia and primate-specific in layer 4 granular neurons. We generated a comprehensive survey of the dlPFC cellular repertoire and its shared and divergent features in anthropoid primates. Description Primate brain diversity Primate brains benefit from an evolutionary specialization of the cortex that supports cognition. Ma et al. tracked transcriptomes from cellular nuclei sampled from human, chimpanzee, macaque, and marmoset brain cortices. The transcriptomes define groups of cell types, some of which are conserved and some that differ between these species. Several human-specific specializations were identified. Expression patterns of more than 900 genes associated with brain disorders revealed a variety of conserved, divergent, group-specific patterns. —PJH Single-cell transcriptomics reveals prefrontal cell taxonomy and species differences in human, chimpanzee, macaque, and marmoset. INTRODUCTION The dorsolateral prefrontal cortex (dlPFC) lies at the center of high-order cognition and complex social behaviors, which are highly derived traits in anthropoid primates—particularly in humans. RATIONALE The granular dlPFC represents an evolutionary specialization found only in anthropoid primates, and alterations in the molecular and cellular mechanisms underlying its intricate circuitry have been implicated in myriad neuropsychiatric diseases. However, little is known about the full repertoire of cell types in the primate dlPFC and how conserved these cell types are between human and other primate species. RESULTS We generated single-nucleus transcriptome data profiling more than 600,000 nuclei from the dlPFC of adult humans, chimpanzees, rhesus macaques, and common marmosets, thus spanning major primate phylogenetic groups. To study regulatory mechanisms underlying human-specific divergence, we generated single-nucleus multiome data (snATAC-seq and snRNA-seq) profiling the human dlPFC. Through cell clustering, marker gene expression, and integration with published multimodal and multispecies data we defined three levels of hierarchically organized taxonomy of transcriptomically defined neuronal, glial, and non-neural cell types in the four species, including four major cell classes, 29 subclasses and 114 subtypes. Most cell subtypes were conserved across the four species but we unraveled prominent species differences both at the molecular and cellular levels. We identified five cell subtypes detected in only a subset of species, including a layer (L) 2-3 intratelencephalic subtype absent in marmosets, an inhibitory neuron subtype exclusive to marmosets, and a microglial subtype detected only in humans. Cross-species comparisons of cell type proportions showed that L2-3 intratelencephalic neurons underwent substantial expansion in humans compared with other species as well as in Catarrhini as compared with marmosets. Gene expression entropy analysis revealed more transcriptomic heterogeneity among L2-3 intratelencephalic neurons in Catarrhini compared to marmosets. These results confirm and extend theories of primate cortical expansion. Within homologous cell subtypes across species, we identified prominent molecular changes. These are characterized by loss of expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine (including dopamine) biosynthesis, in the inhibitory neurons of chimpanzees that are homologous to TH-expressing inhibitory neurons in the other species studied. Among TH-expressing homologous cell subtypes in humans, macaques, and marmosets, we identified a human-specific posttranscriptional switch between the neuropeptide SST and TH, and the human-specific expression of genes involved in dopaminergic function. Through transcriptomic comparisons across the four primate species and immunohistochemistry across 51 mammal species, we found that the neuropsychiatric risk gene FOXP2 exhibited human-specific expression in microglia and primate-specific expression in L4 excitatory neurons. By integrating chromatin accessibility and gene coexpression, we identified cis-regulatory elements regulating FOXP2 expression and constructed FOXP2 regulatory networks including downstream targets mirroring the cell type- and species-specific FOXP2 expression patterns. CONCLUSION We produced a transcriptomic catalog of the primate dlPFC cell types, complemented with epigenomic characterization in the human dlPFC. Our analyses delineated cell type homology and transcriptomic conservation across species and identified species divergence at the molecular and cellular levels, as well as potential epigenomic mechanisms underlying these differences. Shared and species-divergent features were implicated in biological pathways and neuropsychiatric diseases. Our data may serve as a resource for future studies on prefrontal cortex function and disease. Transcriptomic taxonomy of the dlPFC in four anthropoid primates. (Top left) Homologous regions of the dlPFC dissected for snRNA-seq and sn-multiome analyses. (Top right) 114 hierarchically organized (dendrogram) transcriptomically defined cell subtypes distributed across the four species (bar plots; same color code as in the top left panel), with species-specific variations highlighted. (Bottom) Notable molecular changes across species featured by species-specific FOXP2 expression and the human-specific posttranscriptional switching between SST and TH.

Molecular and cellular evolution of the primate dorsolateral prefrontal cortex

The mammalian brain is composed of millions to billions of cells that are organized into numerous cell types with specific spatial distribution patterns and structural and functional properties. An essential step towards understanding brain function is to obtain a parts list, i.e., a catalog of cell types, of the brain. Here, we report a comprehensive and high-resolution transcriptomic and spatial cell type atlas for the whole adult mouse brain. The cell type atlas was created based on the combination of two single-cell-level, whole-brain-scale datasets: a single- cell RNA-sequencing (scRNA-seq) dataset of ∼7 million cells profiled, and a spatially resolved transcriptomic dataset of ∼4.3 million cells using MERFISH. The atlas is hierarchically organized into five nested levels of classification: 7 divisions, 32 classes, 306 subclasses, 1,045 supertypes and 5,200 clusters. We systematically analyzed the neuronal, non-neuronal, and immature neuronal cell types across the brain and identified a high degree of correspondence between transcriptomic identity and spatial specificity for each cell type. The results reveal unique features of cell type organization in different brain regions, in particular, a dichotomy between the dorsal and ventral parts of the brain: the dorsal part contains relatively fewer yet highly divergent neuronal types, whereas the ventral part contains more numerous neuronal types that are more closely related to each other. We also systematically characterized cell-type specific expression of neurotransmitters, neuropeptides, and transcription factors. The study uncovered extraordinary diversity and heterogeneity in neurotransmitter and neuropeptide expression and co-expression patterns in different cell types across the brain, suggesting they mediate a myriad of modes of intercellular communications. Finally, we found that transcription factors are major determinants of cell type classification in the adult mouse brain and identified a combinatorial transcription factor code that defines cell types across all parts of the brain. The whole-mouse-brain transcriptomic and spatial cell type atlas establishes a benchmark reference atlas and a foundational resource for deep and integrative investigations of cell type and circuit function, development, and evolution of the mammalian brain.

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A high-resolution transcriptomic and spatial atlas of cell types in the whole mouse brain

In mammalian brains, tens of millions to billions of cells form complex interaction networks to enable a wide range of functions. The enormous diversity and intricate organization of cells in the brain have so far hindered our understanding of the molecular and cellular basis of its functions. Recent advances in spatially resolved single-cell transcriptomics have allowed systematic mapping of the spatial organization of molecularly defined cell types in complex tissues1–3. However, these approaches have only been applied to a few brain regions1–11 and a comprehensive cell atlas of the whole brain is still missing. Here, we imaged a panel of >1,100 genes in ∼8 million cells across the entire adult mouse brain using multiplexed error-robust fluorescence in situ hybridization (MERFISH)12 and performed spatially resolved, single-cell expression profiling at the whole-transcriptome scale by integrating MERFISH and single-cell RNA-sequencing (scRNA-seq) data. Using this approach, we generated a comprehensive cell atlas of >5,000 transcriptionally distinct cell clusters, belonging to ∼300 major cell types, in the whole mouse brain with high molecular and spatial resolution. Registration of the MERFISH images to the common coordinate framework (CCF) of the mouse brain further allowed systematic quantifications of the cell composition and organization in individual brain regions defined in the CCF. We further identified spatial modules characterized by distinct cell-type compositions and spatial gradients featuring gradual changes in the gene-expression profiles of cells. Finally, this high-resolution spatial map of cells, with a transcriptome-wide expression profile associated with each cell, allowed us to infer cell-type-specific interactions between several hundred pairs of molecularly defined cell types and predict potential molecular (ligand-receptor) basis and functional implications of these cell-cell interactions. These results provide rich insights into the molecular and cellular architecture of the brain and a valuable resource for future functional investigations of neural circuits and their dysfunction in diseases.

https://www.biorxiv.org/content/biorxiv/early/2023/03/07/2023.03.06.531348.full.pdf

A molecularly defined and spatially resolved cell atlas of the whole mouse brain

Humans have unique cognitive abilities among primates, including language, but their molecular, cellular, and circuit substrates are poorly understood. We used comparative single nucleus transcriptomics in adult humans, chimpanzees, gorillas, rhesus macaques, and common marmosets from the middle temporal gyrus (MTG) to understand human-specific features of cellular and molecular organization. Human, chimpanzee, and gorilla MTG showed highly similar cell type composition and laminar organization, and a large shift in proportions of deep layer intratelencephalic-projecting neurons compared to macaque and marmoset. Species differences in gene expression generally mirrored evolutionary distance and were seen in all cell types, although chimpanzees were more similar to gorillas than humans, consistent with faster divergence along the human lineage. Microglia, astrocytes, and oligodendrocytes showed accelerated gene expression changes compared to neurons or oligodendrocyte precursor cells, indicating either relaxed evolutionary constraints or positive selection in these cell types. Only a few hundred genes showed human-specific patterning in all or specific cell types, and were significantly enriched near human accelerated regions (HARs) and conserved deletions (hCONDELS) and in cell adhesion and intercellular signaling pathways. These results suggest that relatively few cellular and molecular changes uniquely define adult human cortical structure, particularly by affecting circuit connectivity and glial cell function.

https://www.biorxiv.org/content/biorxiv/early/2022/09/19/2022.09.19.508480.full.pdf

Comparative transcriptomics reveals human-specific cortical features

The common marmoset as a model of neurodegeneration

Early telencephalic development involves patterning of the distinct regions and fate specification of the neural stem cells (NSCs). These processes, mainly characterized in rodents, remain elusive in primates and thus our understanding of conserved and species-specific features. Here, we profiled 761,529 single-cell transcriptomes from multiple regions of the prenatal macaque telencephalon. We defined the molecular programs of the early organizing centers and their cross-talk with NSCs, finding primate-biased signaling active in the antero-ventral telencephalon. Regional transcriptomic divergences were evident at early states of neocortical NSC progression and in differentiated neurons and astrocytes, more than in intermediate transitions. Finally, we show that neuropsychiatric disease- and brain cancer-risk genes have putative early roles in the telencephalic organizers’ activity and across cortical NSC progression. One-Sentence Summary Single-cell transcriptomics reveals molecular logics of arealization and neural stem cell fate specification in developing macaque brain

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Xoel Mato-Blanco

Papers

Molecular and cellular evolution of the primate dorsolateral prefrontal cortex

Molecular programs of regional specification and neural stem cell fate progression in developing macaque telencephalon